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Trang 1ACS Synthetic Biology is published by the American Chemical Society 1155 Sixteenth Street N.W., Washington, DC 20036
Lara Sioban Small, Marc Bruning, Andrew Thomson, Aimee L Boyle, Roberta Davies, Paul
M G Curmi, Nancy R Forde, Heiner Linke, Derek N Woolfson, and Elizabeth Bromley
ACS Synth Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00037 • Publication Date (Web): 21 Feb 2017
Downloaded from http://pubs.acs.org on February 23, 2017
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Trang 2Construction of a chassis for a tripartite protein-based molecular motor
Authors:
Lara S R Small1, Marc Bruning2, Andrew R Thomson3 , Aimee L Boyle4, Roberta B Davies5, Paul M
G Curmi6, Nancy R Forde7, Heiner Linke8, Derek N Woolfson29†, Elizabeth H C Bromley1 *
*Corresponding Author e.h.c.bromley@durham.ac.uk
Affiliations
1 Department of Physics, Durham University, Durham, UK
2 School of Chemistry, Bristol University, Bristol, UK
3 School of Chemistry, University of Glasgow, UK
4 Faculty of Science, Leiden Institute of Chemistry, Netherlands
5 Structural Biology Laboratory, Victor Chang Cardiac Research Institute, Darlinghurst, Australia
6 School of Physics, University of New South Wales, Australia
7 Department of Physics, Simon Fraser University, Burnaby, Canada
8 Department of Physics, Lund University, Lund, Sweden
9 School of Biochemistry, University of Bristol, UK
† BrisSynBio, University of Bristol, UK
Abstract
Improving our understanding of biological motors, both to fully comprehend their activities in vital processes, and to exploit their impressive abilities for use in bionanotechnology, is highly desirable One means of understanding these systems is through the production of synthetic molecular motors We demonstrate the use of orthogonal coiled-coil dimers (including both parallel and anti-parallel coiled-coils) as a hub for linking other components of a previously described synthetic molecular motor, the Tumbleweed We use circular dichroism, analytical ultracentrifugation,
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Trang 3dynamic light scattering and disulfide rearrangement studies to demonstrate the ability of this six-peptide set to form the structure designed for the Tumbleweed motor The successful formation of a suitable hub structure is both a test of the transferability of design rules for protein folding as well as
an important step in the production of a synthetic protein-based molecular motor
Introduction
Protein-based molecular machines are a vital component of living cells and carry out diverse functions including the transportation of numerous cargoes, muscle contraction and cell division 1, 2 Biological motors are highly complex, regulated machines, making them challenging to study but also a motivating target for use in bionanotechnology 3, 4
Within the field of synthetic biology, the bottom-up approach 5, 6 aims to start with simplified, well-characterised, components and modify or extend their functionality, in this case to produce novel nano-scale motors A key design challenge in this area is the level of reproducibility and transferability of components from one context to another, and determining the ability of components to be combined without unexpected interactions occurring is important
Entirely synthetic, biologically-inspired motors have been designed with varying levels of ability and mimicry, including small molecule machines such as switches, shuttles, and even a robotic arm7, 8 Using more biological starting materials, there has also been significant progress in the field of autonomous DNA motors 9, 10 and hybrid DNA-protein motors11 Neither of these areas, however, utilise the same set of initial building blocks as nature: amino acids The field of synthetic protein motors is far less developed due to the higher complexity of amino acid interactions There are, however, several examples of combining peptides with small molecules to create synthetic machines12-14 and structures such as a cyclic peptide-based nanopore15, but there are, to our knowledge, no peptide or protein-based synthetic motors that avoid the use of existing protein motor domains
This paper describes progress in the realisation of a series of previously published motor designs, including the Tumbleweed and SKIP motors16, 17
These motors are designed to undergo directed motion through rectified diffusion using ligand-controlled binding of their ‘feet’ to a DNA track A peptide `hub' is proposed 16
to self-assemble, connect and organise E Coli protein based ‘feet’ with
the ability to bind orthogonal DNA sequences (in the presence of orthogonal ligands) to form the motor molecule (see Figure 1) Construction of a suitable DNA track 18
and microfluidic device19, 20
have since been demonstrated, along with further simulation studies21, 22 The proposed hub is composed of three peptide sequences, each of which contains two coiled-coil domains23 , with each domain programmed to assemble with a specified partner to produce the final configuration (see Fig 1) Coiled-coil domains have been selected for this application, primarily due
to their ability undergo programmed assembly, however the rigidity of the folded domain is also desirable in reducing the probability of backward stepping and hence improving the efficiency of the final motor The use of coiled-coils in this way is not straight forward as individual coiled-coils sequences have often been found to interact with many other potential partners rather than interacting only with their biologically relevant partner24, 25
It has also been documented in other areas of synthetic biology that once the complexity of synthetic systems increases, unintended cross-talk between components can occur26, 27
In order to design the high fidelity sequences required in this application we must therefore draw on the wealth of information now available on the folding and interactions of coiled coils23, 28, 29 The coiled-coil nanostructure field has advanced on many fronts in recent years including the use of biological coiled-coil domains in novel combinations30, 31, the design of self-assembling peptides from
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Trang 4first principles32-34 and the use of computational design tools35-37 From these works we anticipate the need to use a heptad repeat of hydrophobic residues with polar insertions used to specify the orientation of helix interactions Given our peptides will each contain two coiled-coil domains, we must consider the formation of intra-molecular hairpins to be a primary route to misfolding and use the combination of charged amino acids and polar insertions to the hydrophobic core to specifically design against this eventuality in each peptide In addition to the coiled-coil oligomerization information, knowledge on how to link domains successfully must be extracted from recent studies
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and applied in order to provide sufficient flexibility in between domains to allow folding
In this work we have designed, synthesized and characterized a linked system of three coiled coils, suitable for the formation of a hub for the Tumbleweed motor The design in this work is presented
as a proof of principle that the combined knowledge within the field can be successfully applied to a range of new applications requiring the ordered co-localisation of multiple functional components, including the design of hubs for protein based synthetic molecular motors
Figure 1 The `Y' Configuration of the Tumbleweed Hub (adapted from reference [21]) The peptide hub termini are indicated, showing the parallel coiled coils p1,p2 and p3,p4, and the antiparallel coiled coil p5,p6 To form a hub, coiled-coil pairs (p1,p2), (p3,p4) and (p5,p6) need to preferentially interact, while p1 and p6, p2 and p3, and p4 and p5 are covalently linked, to ensure that all of the coiled coils are connected to form one
structure The non-helical central ribbons represent the residues at one end of every peptide which are designed not to contribute to a coiled-coil region
Results and Discussion
Table 1:
6 nm
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Trang 5Using the design principles laid out previously 38, 40 six peptides were designed to form three dimeric coiled coils: (p1,p2), (p3,p4) and (p5,p6) Polar residues are included in various hydrophobic core positions to prevent staggered assembly and electrostatic interactions are optimised to produce the desired dimeric pairings using an algorithm reported previously40 All contain a tyrosine residue for
UV absorption concentration measurements, a single cysteine residue for disulfide bond formation and a linker of glycine and asparagine intended to form a flexible region outside the coiled-coil
domains on one terminus
The sequences of the six peptides labelled in Figure 1 are shown in Table 1 These were synthesised using solid phase peptide synthesis and purified with high performance liquid chromatography
Several facts must be established in order to confirm the success of the design principles and demonstrate that when all six peptides are mixed, the three required dimeric coiled-coil structures are produced Firstly we must demonstrate that the designed peptide pairs interact with each other
Secondly we must show that the structures formed by these pairs are dimeric in nature Finally we must find that the three designed pairs are produced preferentially over other possible oligomers
The secondary structures of the peptides and two-peptide combinations were studied with circular dichroism (CD) The individual peptides displayed varying levels of helicity (see Fig S1a) All 15 two-peptide mixtures were made, and measured The CD spectra of the three specified dimers, alongside the spectra of the component peptides, are shown in Figure 2a, 2b and 2c All three pairs indicate the presence of heterooligomeric interactions, as the measured spectra of the mixtures show greater helicity than those predicted from the sum of the individual component peptide spectra The data from the other 12 possible combinations of two hub peptides also demonstrate various levels
of heterooligomeric interactions to be present These data show that the specified pairs can be formed To determine whether the desired pairs are formed preferentially to other interactions, all six peptides were mixed together The CD spectrum of this mixture is shown in Figure 2d This has been compared to predicted spectra for six non-interacting species, and to a sample comprising only
of the three designed pairs As expected, the combined spectrum is highly similar to that predicted under the assumption that no further interactions are produced when the designed pairs are mixed
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Trang 6Figure 2
CD spectra of the three mixtures of the designed pairs of peptides: (a) p1 & p2, (b) p3 & p4 and (c), p5 & p6 (shown in green) Each figure also shows the individual peptide spectra (red and blue, in numerical order), and the theoretical signal for a mixture of the two without interaction (a combination of the two single peptide signals, shown in purple) (d) CD spectrum of all six hub peptides mixed together (green), compared with the theoretical spectrum predicted from the individual spectra (shown in magenta), and from the designed pair spectra (shown in purple) Each sample had a concentration of 20 µM per peptide, in PBS buffer (pH 7.4), measured at 20 °C Dotted lines represent the wavelength cutoff below which data cannot
be reliably obtained, as dictated by the CD instrument
Analytical ultracentrifugation (AUC) measurements of the designed coiled-coil pairs (Table 2) gave molecular weights consistent with dimer formation, and inconsistent with monomers or oligomers
of a higher order than dimers (see S.I Tables S1 and S2 for further details)
Table 2
Likewise, dynamic light scattering (DLS) measurements of the designed coiled-coil pairs each exhibited species with 3-5 nm hydrodynamic diameters, consistent with the hypothesis of the formation of discrete coiled-coil oligomers, and inconsistent with the formation of fibres (see S I Fig S2)
In order to demonstrate the fidelity of the interactions and to confirm the utility of the peptides in organising the motor hub, pairs of the peptides were covalently linked using a disulfide bond between terminal cysteine residues, and the disulfide-bonded peptides, p6-1, p3-2 and p4-5 were produced and characterized
The CD spectra of the disulfide-bonded peptides reveal little about the system as all of the collected spectra show high levels of helicity (see SI Fig S1b) This is expected for a system where the peptides have been designed to form coiled-coils and have been co-localised by the disulfide tethering causing conditions of high local concentration and hence non-specific coiled coil formation
AUC results are more informative about the system's interactions (see Table 3) The single disulfide-bonded peptides have the expected masses (The higher than expected value found for 2-3
nonetheless corresponds well to a single species fit, and the mass found is far closer to the mass of a single disulfide-bonded peptide than to a pair.) To study the specificity of coiled-coil interactions, the disulfide-bonded peptides were mixed pairwise and allowed to associate The mixtures of pairs of disulfide-bonded peptides exhibit single species behaviour, indicative of the desired specificity of interaction Furthermore this species in each case has a mass consistent with the interaction of two disulfide-bonded peptides The mixture of all three disulfide-bonded peptides gives a mass
consistent with the expected mass of 20,248 Da, again supportive of our design criteria
Table 3
Molecular Weight / Da
Measured Molecular Weight / Da
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Trang 7p6-1, p2-3 13566 16000 ± 1000
DLS data of the individual disulfide-bonded peptides gave hydrodynamic diameters of 3-4 nm, while DLS data of pairs of the disulfide-bonded peptides, and of all three disulfide-bonded peptides mixed, showed a shift to higher values, around 5 nm (see S.I Fig S3) The change in size between the single and pairs of bonded peptides indicates that there is interaction between the disulfide-bonded peptides The fact that the size of the three-component mixture is similar to that of the pairs
of disulfide-bonded peptides is supportive that a structure such as that shown in Figure 1 is formed, and not a lengthy fibre
Figure 3
If the designed dimer peptides preferentially interact, when allowed to rearrange, the disulfide bonds should switch to between the desired pairs, with the species present in the system moving from p6-1, p3-2 and p4-5 to p1-2, p3-4 and p5-6
To further verify that the interactions occurring in the final structure are due to the three specified peptide pairs, we carried out disulfide exchange experiments on the linked peptides We measured the system of three disulfide-bonded peptides, and then changed the system's redox potential, using reduced glutathione, to allow the disulfide bonds to rearrange If there are preferred interactions, the disulfide bonds formed when rearrangement is possible should reflect these preferences (see Fig 3) We measured the species in each of these samples using MALDI-ToF mass spectrometry, and the results are shown in Figure 4 Prior to rearrangement, the species present in the system are the disulfide-bonded peptides produced in the laboratory (see Figure 4a) Once the disulfide bonds are given the opportunity to rearrange (see Fig 4b), the species present change Disulfide-bonded versions of all three of the designed pairs (p1-2, p3-4, p6-5) produce the dominant peaks, along with
a fourth peak attributed to the starting peptide 2-3 (that is likely due to a failure to produce a precise 1:1:1 ratio of the starting species) Regardless of the presence of this peak, it is clear that the three designed pairs interact when given the opportunity, while none of the other possible
combinations not present in the initial system form during rearrangement (see S.I table S3 for details of peak assignments)
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Trang 8Figure 4 MALDI Spectra of Glutathione Experiments The initial spectrum has prominent peaks due to the p4-5 (MW
6682 Da) and p3-2 (MW 7095 Da) species, and smaller peaks due to deletions and the p6-1 peptide (MW
6470 Da) After rearrangement, the p4-5 and p6-1 species are no longer seen, while the p6-5 (MW 5814 Da), p1-2 (MW 7039 Da) and p3-4 (MW 7395 Da) species have appeared All of the trace peaks correlate with impurities in the initial samples and do not correlate with possible rearrangements
This combined information from CD, DLS, AUC and disulfide rearrangement experiments indicates that the hub construction should indeed be possible from mixing covalently linked species of peptides p1 and p6, p2 and p3, and p4 and p5, driven by coiled-coil interactions As such, this has been a successful test of the hypothesis that the knowledge within the peptide design field is now sufficient for the design of self-assembling hubs for diverse applications, including the co-localisation
of other motor components
Future extensions of this work could investigate whether it is possible to combine this synthetic biology approach to structure design with active means of actuating the structure Using the observed ability of peptides41-43
to switch conformation in different conditions could provide a feasible means of adapting this system to undergo conformational changes (and hence changes in
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(b)
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Trang 9movement) in the presence of different stimuli This will take us one step closer to realising designed protein motion
Conclusions
We have demonstrated that a set of six coiled-coil domains can be designed to self-assemble preferentially into three coiled-coil heterodimers We have shown that by pairing the domains covalently, a hub structure can be formed This hub can then be used to assembly multiple functional units into the desired 3D configuration We propose using this technology to produce designed protein motor hubs, however, the methodology exploited here may have wider applications in the design and production of self-assembling junctions in other areas of bionanotechnology including the construction of synthetic cages or other nanoscale devices
Materials and Methods
Amino acids and PyBOP were purchased from AGTC Bioproducts Ltd DMF was purchased from Rathburn Chemicals and AGTC Bioproducts Ltd Rink amide MBHA resin was purchased from Novabiochem Other synthesis materials, including Aldrithiol-2, were purchased from Sigma Aldrich
HPLC grade acetonitrile was purchased from Fisher Scientific
Peptides were synthesised using SPPS using a CEM Liberty 1 peptide synthesiser under standard conditions HPLC purification was carried out using linear water to acetonitrile gradients, with a Perkin Elmer 785A detector and Series 200 LC pump, and a C18 column MALDI-ToF experiments were carried out using a Bruker Daltonics Autoflex II ToF/ToF machine, and
α-cyano-4-hydroxycinnamic acid matrix
To form each disulfide-bonded peptide, one of the contributing peptides (dissolved in water) was reacted with a tenfold excess of Aldrithiol-2 (in acetonitrile), and the products were separated by HPLC The peptide product was then reacted with the other contributing peptide, and through displacement formed a disulfide-bonded peptide The waste products of this reaction were then removed using HPLC
Concentrations were determined using UV/vis absorption spectroscopy and the tyrosine absorption centred around 276nm: εTyr = 1280 cm-1 mol-1 With the exception of UV/vis spectra for the disulfide exchange experiments, which used a Thermo Scientific Nanodrop 1000, spectra were taken using a Unicam UV2 spectrometer
CD spectroscopy was carried out using a JASCO J-1500 spectropolarimeter with mini circulation bath and peltier stage, using a 1mm quartz cuvette Samples were made at a concentration of 20 μM per peptide, in PBS buffer
AUC sedimentation equilibrium data was collected at 20 °C in PBS buffer using a Beckman Optima XL-I analytical ultracentrifuge with an An-60 Ti rotor Temperatures: The partial specific volume for
each of the peptides/peptide mixtures, and the solvent density, were calculated using Sednterp
DLS measurements were taken on a Malvern Zetasizer µV using Sarstedt disposable transparent cuvettes Samples were made at a concentration of 100 µM per peptide, in PBS buffer
TFA in water solutions (pH 2) were found to quench the disulfide exchange reaction, and hence this was used to both prevent rearrangement of the initial species prior to MALDI analysis, and to quench the system after rearrangement was encouraged in a second sample Each of the disulfide-bonded peptides, in water solution, was added to both a solution of TFA in water (4 µl TFA in 15ml
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Trang 10water, pH 2) and to a PBS buffer solution containing reduced glutathione After ten minutes, TFA in water was added to the glutathione-containing solution to quench the reaction These two solutions were then studied using MALDI-ToF mass spectrometry
Acknowledgements
The authors thank EPSRC for funding of LSRS through a Doctoral Studentship (EP/P505488/1) and Doctoral Prize (EP/M507854/1) ART and DNW are supported by the ERC (340764) and DNW is a Royal Society Wolfson Research Merit Award holder (WM140008)
1 Vale, R D (2003) The Molecular Motor Toolbox for Intracellular Transport, Cell 112,
467-480
2 Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter., P (2002) Molecular
Biology of the Cell, 4th Edition, 4 ed., Garland Science, New York
Science 317, 333-336
5 Bromley, E H C., Channon, K., Moutevelis, E., and Woolfson, D N (2008) Peptide and
protein building blocks for synthetic biology: From programming biomolecules to
self-organized biomolecular systems, Acs Chemical Biology 3, 38-50
biomolecular design and engineering, Current Opinion in Structural Biology 18, 491-498
7 Erbas-Cakmak, S., Leigh, D A., McTernan, C T., and Nussbaumer, A L (2015) Artificial
Molecular Machines, Chem Rev 115, 10081-10206
8 Kassem, S., Lee, A T L., Leigh, D A., Markevicius, A., and Sola, J (2016) Pick-up, transport
and release of a molecular cargo using a small-molecule robotic arm, Nat Chem 8, 138-143
9 Chen, Y J., Groves, B., Muscat, R A., and Seelig, G (2015) DNA nanotechnology from the
test tube to the cell, Nat Nanotechnol 10, 748-760
10 Pan, J., Li, F R., Cha, T G., Chen, H R., and Choi, J H (2015) Recent progress on DNA based
walkers, Curr Opin Biotechnol 34, 56-64
11 Lund, K., Manzo, A J., Dabby, N., Michelotti, N., Johnson-Buck, A., Nangreave, J., Taylor, S.,
Pei, R J., Stojanovic, M N., Walter, N G., et al (2010) Molecular robots guided by prescriptive landscapes, Nature 465, 206-210
12 Ikezoe, Y., Fang, J., Wasik, T L., Shi, M L., Uemura, T., Kitagawa, S., and Matsui, H (2015)
Peptide-Metal Organic Framework Swimmers that Direct the Motion toward Chemical
Targets, Nano Lett 15, 4019-4023
of a metal-organic framework powered by reorganization of self-assembled peptides at
interfaces, Nat Mater 11, 1081-1085
14 Nagatsugi, F., Takahashi, Y., Kobayashi, M., Kuwahara, S., Kusano, S., Chikuni, T., Hagihara,
S., and Harada, N (2013) Synthesis of peptide-conjugated light-driven molecular motors and
evaluation of their DNA-binding properties, Mol Biosyst 9, 969-973
Chemical Society Reviews
16 Bromley, E H C., Kuwada, N J., Zuckermann, M J., Donadini, R., Samii, L., Blab, G A.,
Gemmen, G J., Lopez, B J., Curmi, P M G., Forde, N R., et al (2009) The Tumbleweed: towards a synthetic protein motor, Hfsp J 3, 204-212
17 Zuckermann, M J., Angstmann, C N., Schmitt, R., Blab, G A., Bromley, E H C., Forde, N R.,
Linke, H., and Curmi, P M G (2015) Motor properties from persistence: a linear molecular
walker lacking spatial and temporal asymmetry, New Journal of Physics 17, 13
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